我们感谢孟博士林青女士远东的技术援助。这项工作是从美国国立卫生研究院HD053767（转包NLW），由授出从生理和副校长办公室的研究，美国加州洛杉矶大学（NLW）部的资金支持。

1。成人脑解剖鳉<ol><li>麻醉成年男性或女性（ 图1A）浸泡5毫升的MS-222（150毫克/升，pH值7.4），等待一两分钟后，鳃变动之前已经停止斩首。加州洛杉矶大学的机构动物护理和使用委员会批准的所有程序。 </li><li>杀头在盐水鱼的鱼鳃盖的尾端用剪刀在一个直径为60毫米的培养皿。 </li><li>传输鱼头一个直径为35毫米的培养皿中，一半充满了盐水鱼及内衬SYLGARD有抑郁症，它为大脑解剖定位和固定头部。位置和固定头背的一面朝上，昆虫针。 </li><li>小心切断背侧部围绕其周边的​​头骨与细剪刀，轻轻取出镊子，露出大脑完全包括前部的脊髓背侧部分。 </li> &lt;LI&gt;轻轻抬起脊髓细尖镊子和断绝双边结缔组织的细尖的剪刀:颅神经，脊神经，视神经，大脑腹侧神经，嗅觉神经的前部大脑。 </li><li>将充分解剖的大脑在一个充满盐水鱼的新鲜的培养皿中，并在显微镜下仔细检查，以确保没有任何切口或穿刺，全脑是否完好（包括脑垂体）。受损的大脑不用于实验。 </li></ol> 2。在录音室中安装的大脑<ol><li>瞬干胶或其他一些快熔胶用针进入录音室的中心放置一小滴和传播胶约1厘米2。 </li><li>迅速转移大脑和胶水中腹的一面朝上位置。 </li><li>添加1毫升的盐水鱼，使其充满录音室，并涵盖了大脑。不断灌注的大脑与曝气鱼生理盐水至少为200微升/分钟的速率。 </li><li>使用细尖钳子小心地取出脑膜的大脑的表面上的记录区域。 </li></ol> 3。电<ol><li>松散的膜片钳记录动作电位射击<ol><li>玻璃微电极（6-10MΩ）构建的硼硅玻璃吸管（1B150F-4，世界精密仪器）使用电极拉马（ 如萨特的P-87），和背面充满了半路上了松散的膜片录音解决方案电极。 </li><li>使用一个直立的荧光显微镜用冷却的电荷耦合器件（CCD）摄像机，发现GFP-表达TN-GnRH3的神经元通过10倍（ 图1B），然后40倍的水浸泡的目标（ 图1C）。它们存在于嗅球，只是前端脑腹侧表面处或附近。 </li><li>切换到视频显示器提供从显微镜的实时图像定位的TN-GnRH3的神经元集群（ 图1C）。 </li><li>微电极定位入熔池，使尖端的电​​极上方的神经元靶。适用于恒定的轻微的正压力，以便它不被堵塞，并轻轻地接近目标神经元与电极尖端的微电极。 </li><li>检查和监控电压钳模式的使用AxoGraph软件和的放大器Axoclamp 200B与Axograph软件（Axon仪器）的电极电阻。的时候可以看到，电极尖端从视频监视器的表面上的神经元和从的AxoGraph显示的电阻略有变化，释放的正压力，并如果有必要施加轻微的负压力，使一个低电阻的密封件的神经元（&lt;100MΩ）。 </li><li>切换电流钳模式下，连续记录膜电压没有任何电流输入，调整规模电压和如果有必要的记录率。 PowerLab系统软件（的ADInstruments公司）的数据收集和分析。 </li><li>记录基线电活动的正常鱼的盐水任何治疗（ 图2A）的前5分钟左右。浴灌注药物，激素，肽等 。添加到鱼盐水继续上面的速率为200微升/分钟，然后用洗脱期在正常鱼生理盐水5，6，11。 </li></ol></li><li>全细胞记录膜电位 膜片钳全细胞电生理过程是相同的如上述外松补丁录制过程中，从步骤3.1.1 - 3.1.4。然后程序相应的偏离: <ol><li>检查和监控电压钳模式的电极电阻。的时候可以看到，电极尖端从视频监视器的表面上的神经元，并从示波器的电阻变小，释放的正压力和施加一点点的负面压力，使高电阻印章上的神经元（〜3千兆Ω）。轻轻吸口破裂修补膜，你会看到一个突然下降的阻力约120-250MΩ。 </li><li>切换电流钳模式下，连续记录膜电压没有任何电流输入，调整电压和如果有必要的记录速率的规模。 </li><li>记录基线电活动的正常鱼生理盐水任何治疗前，约5分钟，如上述3.1.7中（ 图2B）。 </li></ol></li></ol>

<ol>
	<li>Kah, O., Lethimonier, C., Lareyre, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadotrophin-releasing+hormone+(GnRH)+in+the+animal+kingdom.">Gonadotrophin-releasing hormone (GnRH) in the animal kingdom.</a> J. Soc. Biol. 198 (1), 53-60 (2004).</li><li>Ramakrishnan, S., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+from+conspecifics+alter+electrical+activity+of+gonadotropin-releasing+hormone+neurons+in+the+terminal+nerve+via+visual+signals.">Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (1), R135-R141 (2009).</li><li>Abe, H., Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+neuromodulation+by+a+nonhypophysiotropic+GnRH+system+controlling+motivation+of+reproductive+behavior+in+the+teleost.">Mechanisms of neuromodulation by a nonhypophysiotropic GnRH system controlling motivation of reproductive behavior in the teleost.</a> 57 (6), 665-674 (2011).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodotoxin-resistant+persistent+Na++current+underlying+pacemaker+potentials+of+fish+gonadotrophin-releasing+hormone+neurones.">Tetrodotoxin-resistant persistent Na+ current underlying pacemaker potentials of fish gonadotrophin-releasing hormone neurones.</a> J. Physiol. 482 (Pt. 1), 1-6 (1995).</li><li>Zhao, Y., Wayne, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Kisspeptin1+on+Electrical+Activity+of+an+Extrahypothalamic+Population+of+Gonadotropin-Releasing+Hormone+Neurons+in+Medaka.">Effects of Kisspeptin1 on Electrical Activity of an Extrahypothalamic Population of Gonadotropin-Releasing Hormone Neurons in Medaka.</a> PLoS One. 7 (5), e37909 (2012).</li><li>Wayne, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+electrophysiology+of+gonadotropin-releasing+hormone+neurons+that+express+green+fluorescent+protein+in+the+terminal+nerve+of+transgenic+medaka+(Oryzias+latipes).">Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).</a> Biol. Reprod. 73 (6), 1228-1234 (2005).</li><li>Okubo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forebrain+gonadotropin-releasing+hormone+neuronal+development:+insights+from+transgenic+medaka+and+the+relevance+to+X-linked+Kallmann+syndrome.">Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.</a> Endocrinology. 147 (3), 1076-1084 (2006).</li><li>Okubo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+form+of+gonadotropin-releasing+hormone+in+the+medaka,+Oryzias+latipes.">A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes.</a> Biochem. Biophys. Res. Commun. 276 (1), 298-303 (2000).</li><li>Ramakrishnan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+spontaneous+electrical+activity+during+embryonic+development+of+gonadotropin-releasing+hormone-3+neurons+located+in+the+terminal+nerve+of+transgenic+zebrafish+(Danio+rerio).">Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).</a> Gen. Comp. Endocrinol. 168 (3), 401-407 (2010).</li><li>Abraham, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gonadotropin-releasing+hormone-3+neuron+ablation+in+zebrafish:+effects+on+neurogenesis,+neuronal+migration,+and+reproduction.">Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.</a> Endocrinology. 151 (1), 332-340 (2010).</li><li>Wayne, N. L., Kuwahara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-endorphin+alters+electrical+activity+of+gonadotropin+releasing+hormone+neurons+located+in+the+terminal+nerve+of+the+teleost+medaka+(Oryzias+latipes.">Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes.</a> Gen. Comp. Endocrinol. 150 (1), 41-47 (2007).</li><li>Oka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+types+of+gonadotrophin-releasing+hormone+neurones+and+steroid-sensitive+sexually+dimorphic+kisspeptin+neurones+in+teleosts.">Three types of gonadotrophin-releasing hormone neurones and steroid-sensitive sexually dimorphic kisspeptin neurones in teleosts.</a> J. Neuroendocrinol. 21 (4), 334-338 (2009).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping: An Introductory Guide To Patch Clamp Electrophysiology. , (2003).</li></ol>

什么都没有透露。

激素3:提供了独特的绿色荧光蛋白转基因鱼模型来研究的神经生理机制神经集成中央控制和调节的行为，都直接和间接地参与再现3 8-10。这个模型系统的显着优势之一是，许多GnRH3表达GFP神经元的大脑腹面，可以相对容易获得的神经元电生理记录，不破坏神经回路6，9，11，12（ 图图1B和图1C，图3A和3B）。在这段视频中，我们已经表明了?...

<table><tbody><tr><td>Microscope</td><td>Olympus</td><td></td><td>BX50W (Upright)</td></tr><tr><td>Amplifier</td><td>Axon Instruments</td><td></td><td>Axoclamp 200B </td></tr><tr><td>A-D converter</td><td>Computer Interference Corp.</td><td></td><td>Digidata ITC-18 </td></tr><tr><td>Cooled CCD camera</td><td>PCO Computer Optics</td><td></td><td>Sensicam</td></tr><tr><td>Xenon lamp</td><td>Sutter Instruments Co.</td><td></td><td></td></tr><tr><td>GFP filter set</td><td>Chroma Technologies</td><td></td><td></td></tr><tr><td>Imaging Software</td><td>Intelligent Imaging Innovations</td><td></td><td>Slidebook software </td></tr><tr><td>Electrophysiology Data Acquisition Software</td><td>Axon Instruments</td><td></td><td>Axograph software </td></tr><tr><td>Electrophysiology Data Acquisition Software</td><td>AD Instruments Inc.</td><td></td><td>PowerLab </td></tr><tr><td>Headstage for electrophysiology</td><td>Axon Instruments</td><td></td><td>CV 203BU </td></tr><tr><td>Micromanipulator</td><td>Sutter Instrument Co</td><td>MP-285</td><td></td></tr><tr><td>Recording Chamber Platform</td><td>Warner Instrument Corp.</td><td></td><td>P1 </td></tr><tr><td>Recording Chamber</td><td>Warner Instrument Corp.</td><td></td><td>RC-26G </td></tr><tr><td>Electrode Puller</td><td>Sutter instruments</td><td></td><td>P87 </td></tr><tr><td>Filament for electrode puller</td><td>Sutter Instruments</td><td>FB330B</td><td>3.0 mm wide trough filament</td></tr><tr><td>1.5 mm glass capillaries</td><td>World Precision Instruments</td><td>1B150-4</td><td>Micr&amp;#8211;lectrode for recording</td></tr><tr><td>Syringe</td><td>Becton Dickinson</td><td>309586</td><td>3 ml</td></tr><tr><td>MS-222 </td><td>Sigma</td><td>E10521-10G</td><td>Ethyl 3-aminobenzoate methanesulfonate salt</td></tr><tr><td>Fish saline</td><td></td><td></td><td>mM: 134 NaCl; 2.9 KCl; 2.1 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1.2 MgCl&lt;sub&gt;2&lt;/sub&gt;; 10 HEPES </td></tr><tr><td>Electrode solution (loose-patch) </td><td></td><td></td><td>mM: 150 NaCl; 3.5 KCl; 2.5 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1.3 MgCl&lt;sub&gt;2&lt;/sub&gt;; 10 HEPES; 10 glucose </td></tr><tr><td>Electrode solution (whole-cell patch) </td><td></td><td></td><td>mM: 112.5 K-gluconate; NaCl; 17.5 KCl; 0.5 CaCl&lt;sub&gt;2&lt;/sub&gt;; 1 MgCl&lt;sub&gt;2&lt;/sub&gt;; 5 MgATP; 1 EGTA; 10 HEPES; 1 GTP; 0.1 leupeptin;10 phospho-creatine </td></tr></tbody></table>

recording electrical activity from identified neurons in the intact brain of transgenic fish

了解细胞的生理调节复杂的行为的神经回路是通过使用模型的系统，在该系统中，可以进行这项工作，在一个完整的大脑制备的中枢神经系统的神经电路保持不变大大增强。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑中的绿色荧光蛋白基因的标签。鱼GnRH神经元有多个种群，它们的功能是依赖于他们的位置在大脑和GnRH基因，他们表达1。我们集中示范GnRH3在终端转基因青鳉鱼（ 图1B和C）使用完整的大脑嗅球神经（田纳西州）的神经元位于。研究表明，青鳉的TN-GnRH3神经元是神经调节，中枢神经系统作为信息发射器从外部环境; T嘿，不要直接发挥作用，调节垂体-性腺功能，做知名GNRH1丘脑神经元2，3。进补动作电位射击TN-GnRH3神经元的自发模式的内在属性4-6，频率是调制的同种2和神经肽kisspeptin 1 5的视觉线索。在这段视频中，我们使用了一个稳定的转基因青鳉这TN-GnRH3的神经元表达了含转基因的启动子区域Gnrh3向您展示如何识别神经元，并监察他们在整个大脑的电活动与增强型绿色荧光蛋白7制备例6。

GFP标记的TN-GnRH3神经元从切除大脑的青鳉鱼的双边集群的一个例子示于图1B和图1C。每个集群包含大约8-10 GnRH神经元。自发的神经元活动的目标TN-GnRH3的被记录在电流钳模式（I = 0），与典型的发射率0.5-6赫兹。动作电位射击模式是一个典型的补药或跳动模式，一个相当普通的峰峰间隔。样品的痕迹显示在图2（2A:宽松的补丁; 2B:全细胞）。 ?...

Watch this Scientific Journal Video about Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish at JoVE.com

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

在这段视频中，我们将演示如何记录保留复杂的神经回路在整个大脑的准备，从确定单个神经元的电活动。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑制备一种荧光蛋白基因的标签。

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

recording-electrical-activity-from-identified-neurons-intact-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of California, Los Angeles. 在这段视频中，我们将演示如何记录保留复杂的神经回路在整个大脑的准备，从确定单个神经元的电活动。我们使用转基因鱼中，促性腺激素释放激素（GnRH）的神经元与用于识别完整的大脑制备一种荧光蛋白基因的标签。

视频: Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

生物学

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

自由游泳鱼中可移动的特龙对神经元的无线电生理记录

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Source: Muto, A. et al.,&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/57485">Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae.</a> J. Vis. Exp. (2018)
This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

分析转基因斑马鱼幼虫中猎物诱发的神经元活动

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope
NOTE: If users plan on performing Ca imaging following ablation, use the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Adult Zebrafish Electrophysiology: A Method to Measure the Extracellular Electrical Activity of Neurons In Vivo

Source: Johnston, L., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/51065/" target="_blank">Electrophysiological Recording in the Brain of Intact Adult Zebrafish</a>. &#160;J. Vis. Exp. (2013)
The protocol describes the procedure to perform electrophysiology in adult zebrafish. This method can be used for measuring neural activity in vivo.

成年斑马鱼电生理学:一种测量体内神经元细胞外电活动的方法

Source: Johnston, L., et al. Electrophysiological Recording in the Brain of Intact Adult Zebrafish. &#160;J. Vis. Exp. (2013)
The protocol describes the ...